Continuous filtration by feed circulation

Abstract

A method and system for continuous filtration of liquid feed is provided, wherein multiple quantities of liquid feed samples are fed in series or simultaneously from two or more sources into a circulation tank, and then the liquid feed samples are fed into a filtration system until the total volume of processed product is reached.

Description

Background Technology

[0001] Filtration typically involves removing impurities and other low-molecular-weight substances from a bioproduction sample in which the target molecule is present, such as proteins, peptides, nucleic acids, or antibodies. Other uses of filtration include purification, where the product may be present in the retention or permeate (or both). While filtration can be performed at several stages throughout the bioproduction process, it is often carried out as a preliminary step to remove impurities such as host cell proteins (HCPs) and nucleic acids (NAs), or as a final step to purify and sterilize the final product pool containing the target molecule.

[0002] Filtration is typically performed in a "batch" manner, meaning that a batch of production run is filtered, followed by rinsing or cleaning the filtration system in preparation for the next batch. Filtration can also be performed as a batch-recirculation operation, where a sample volume is repeatedly passed through the filter assembly several times, with the retained stream from the filter assembly recycled back to the feed tank, and fresh buffer solution introduced into the feed tank, if necessary or required, to replenish any volume lost during filtration. Filtration of the sample continues to be repeated until the final processing conditions are met, at which point the product is recovered from the system. Alternatively, a sample volume can travel through a series of single-pass filter assemblies (i.e., in-line filtration), with buffer solution introduced between each filtration stage, if necessary or required. By using a series of single-pass filtration stages, the inflow and outflow of the sample through the filtration system can be continuous. However, because this process is typically stopped for maintenance after each production "batch," the entire process is a batch process. Summary of the Invention

[0003] Filtration methods and systems are provided in which the continuous inflow and outflow of biological production samples (or “liquid feed samples,” “feed samples,” or “products”) can be achieved using batch filtration processes and components. Such methods and systems provide continuous production while requiring a smaller membrane area and / or buffer volume than typically required for in-line filtration. These methods also typically use a smaller footprint, thus saving valuable floor space.

[0004] In one aspect, the present invention relates to a method for continuous filtration, wherein a first quantity of liquid feed sample is delivered from a sample source, a continuous sample source, or a continuous feed stream to a circulation tank (also referred to as a feed tank). The feed stream is then delivered to a filtration system, typically a tangential flow filtration (TFF) system. During the filtration of the first quantity of liquid feed sample, one or more additional quantities of liquid feed sample are delivered in series or parallel (i.e., simultaneously) to the circulation tank to maintain a liquid feed sample level in the circulation tank, thereby ensuring a continuous feed flow to the filtration system. This method can continue until the total product volume has been processed through the filtration system.

[0005] On the other hand, additional liquid feed samples can come from multiple sample sources, such as from several cell culture devices.

[0006] On the other hand, the filtration system is a TFF system, and in some cases, the filtration system is a single-pass tangential flow filtration (SPTFF) system.

[0007] In some respects, liquid feed samples may be concentrated or diluted by the filtration process. In other respects, the feed stream may be concentrated or diluted before or after the filtration process.

[0008] The feed stream is filtered by a filtration process, wherein one or more target molecules (e.g., monoclonal antibodies) constitute a larger percentage of the non-aqueous component of the filtered liquid compared to an unfiltered liquid feed sample. In some aspects, the concentration of one or more target molecules increases by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 1000%, or more compared to an unfiltered liquid feed sample. Similarly, in some aspects, the concentration of one or more impurities (e.g., host cell proteins (HCP) and / or nucleic acids (NA)) decreases compared to an unfiltered liquid feed sample. In some aspects, the concentration of one or more impurities decreases by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more compared to an unfiltered liquid feed sample. In a preferred aspect, due to the filtration process of the present invention, one or more target molecules are concentrated, while the concentration of one or more impurities is reduced, compared to an unfiltered liquid feed sample.

[0009] The filtration method of the present invention is applicable to any liquid feed sample, provided that the filter is compatible with the liquid feed sample. Those skilled in the art can determine the appropriate filter type (e.g., size porosity, membrane material, etc.) for a particular liquid feed sample to be filtered. In one aspect, the liquid feed sample is a cell culture medium from a cell culture vessel, wherein the cell culture contained in the cell culture vessel is engineered to produce biomolecules with therapeutic properties and / or suitable for use in research and development in the fields of biotechnology and medicine.

[0010] Exemplary biomolecules that can be generated from cell cultures (which produce liquid feed samples for the filtration method of the present invention) include, but are not limited to, monoclonal antibodies and other proteins and glycoproteins with known or potential therapeutic properties.

[0011] In one aspect, the present invention provides a method for filtering a liquid feed sample, the method comprising: (a) conveying a first amount of liquid feed sample from a first sample source to a circulation tank; (b) simultaneously or in series with the first amount of liquid feed sample, conveying a second amount of liquid feed sample from a second sample source to the circulation tank; (c) while a suitable amount of liquid feed sample is collected in the circulation tank, conveying a continuous feed stream of the liquid feed sample from the circulation tank to a tangential flow filtration (TFF) system to filter the continuous feed stream of the liquid feed sample; (d) during the filtration of the liquid feed sample, conveying one or more additional amounts of liquid feed sample to the circulation tank to maintain a continuous feed stream of liquid feed sample to the TFF system; and (e) continuing to supply liquid feed sample to the circulation tank until the total product volume is processed by the TFF system.

[0012] In another aspect, the present invention provides a method for filtering a liquid feed sample, the method comprising: (a) conveying a first amount of liquid feed sample from a first sample source to a circulation tank; (b) while a suitable amount of liquid feed sample is collected in the circulation tank, conveying a continuous feed stream of the liquid feed sample from the circulation tank to a tangential flow filtration (TFF) system to filter the continuous feed stream of the liquid feed sample; (c) during the filtration of the liquid feed sample, conveying one or more additional amounts of liquid feed sample to the circulation tank to maintain a continuous feed stream of the liquid feed sample to the TFF system; and (d) continuing to supply liquid feed sample to the circulation tank until the total product volume is processed by the TFF system.

[0013] In another aspect, the invention specifies that multiple quantities of liquid feed samples come from one or more different sample sources.

[0014] In another aspect, the present invention specifies that the TFF system is a single-pass tangential flow filtration (SPTFF) system.

[0015] In another aspect, the present invention provides that the liquid feed sample is concentrated due to filtration.

[0016] In another aspect, the invention specifies that the liquid feed sample is concentrated before being transported to the circulation tank.

[0017] In another aspect, the present invention specifies that the liquid feed sample is a cell culture medium.

[0018] In another aspect, the present invention specifies that the liquid feed sample contains a monoclonal antibody.

[0019] In another aspect, the invention specifies that the liquid feed sample is diluted before being transported to the circulation tank.

[0020] In another aspect, the invention specifies filtration as percolation, and the method further includes a percolation buffer tank in fluid communication with the feed stream.

[0021] In another aspect, the invention specifies that the method further includes a storage tank between the circulation tank and the TFF system.

[0022] In another aspect, the invention specifies that the method further includes rinsing the TFF system after the total volume of processed product has been reached.

[0023] In some implementations, the continuous filtration method includes a tangential flow filtration (TFF) step. The continuous filtration method can be, for example, a constant volume filtration method or an optimal volume filtration method.

[0024] In a further embodiment, the liquid feed sample can be concentrated before being conveyed to the circulation tank, for example, by a tangential flow filtration (TFF) method or a single-pass tangential flow filtration (SPTFF) method. The method may optionally further include alternately conveying the retentates from the first and second volumes of the liquid feed sample to a storage tank before conveying the retentates to subsequent filtration assemblies. The retentates produced by each volume filtration can be alternately filtered by filtration assemblies, for example, by single-pass tangential flow filtration (SPTFF), until the total product volume is recovered. The method may further include cleaning the circulation tank after the method is completed. The filtration assemblies can be cleaned and / or rinsed after the method is completed.

[0025] In some embodiments, the filtration assembly includes a tangential flow filtration (TFF) assembly. The system may further include a single-pass tangential flow filtration (SPTFF) assembly upstream and / or downstream of the circulation tank to concentrate the sample before and / or after filtration. The system may also include a storage tank or device for containing a surge volume upstream or downstream of the circulation tank.

[0026] In another embodiment, the system includes a percolation buffer tank. The percolation pump may be in fluid communication with both the percolation buffer tank and the circulation tank, or may be in fluid communication with a fluid conduit configured to direct the retention to the circulation tank. The system may also include multiple percolation buffer tanks, which may contain the same or different buffer solutions.

[0027] In another embodiment, the fluid contact flow path of the system is sterilized or disinfected to establish a closed system and includes sterilized / disinfected components that have been sterilized / disinfected by gamma irradiation, ETO, NaOH or other methods and are aseptically connected using pipe welding or aseptic connectors, and are connected to a continuous percolation system using sterile filters on the inlet and outlet lines for controlling bioload. Attached Figure Description

[0028] The foregoing will become apparent from the following more detailed description of exemplary embodiments as shown in the accompanying drawings, in which similar reference characters refer to the same parts in all the different views. These drawings are not necessarily drawn to scale, but rather focus on illustrating the embodiments.

[0029] Figure 1 This is a schematic diagram of the continuous ultrafiltration system of the present invention, which consists of very few components while still achieving a continuous filtration process.

[0030] Figure 2 To illustrate the difference in total membrane area (m²) between the system and method of the present invention and prior art discontinuous filtration methods (batch mode) 2 A diagram illustrating the differences in demand.

[0031] Figure 3 To demonstrate the difference in total membrane loading (kg / m³) between the system and method of the present invention and prior art discontinuous filtration methods (batch mode), 2 A diagram illustrating the differences in demand.

[0032] Figure 4 A diagram illustrating the difference in total tank volume (L) requirement between the system and method of the present invention and prior art discontinuous filtration methods (batch mode).

[0033] Figure 5 A table comparing the various parameter requirements between TFF batch mode and continuous ultrafiltration (UF / DF).

[0034] Figure 6 A table showing a comparison of reagent consumption (NaOH, WFI (water for injection), and buffer) between TFF batch mode and continuous ultrafiltration (UF / DF). Detailed Implementation

[0035] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0036] Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” used herein include the plural.

[0037] As used herein, "continuous filtration method" or "CFP" refers to a method in which feed streams from more than one source are combined in series or parallel before being directed to a filter assembly. CFP may also be referred to as a "continuous filtration system" or "filtration system".

[0038] As used herein, “continuous filtering component” or “CFA” refers to a filtering component configured for use in a continuous method and comprising one or more filtering modules (e.g., a TFF component).

[0039] The term "continuous percolation method" or "CDF method" as used in this article refers to a method in which buffer exchange of liquid feed samples is performed by adding buffer and removing percolate (with a minimum uninterrupted flow into and out of the percolation unit).

[0040] As used herein, "continuous percolation assembly" or "CDF assembly" refers to a filtration assembly configured for use in a continuous percolation method and comprising one or more filtration modules (e.g., a TFF assembly). A "filtration assembly" refers to an assembly comprising one or more filtration units or modules, such as (but not limited to) cartridge filters, spiral wound filters, capsule filters, or hollow fiber filters. A filtration assembly may include one or more filtration units or modules operating in series or in parallel. Series operation refers to a module in which the residue of a preceding module becomes the feed for a subsequent module.

[0041] The terms “TFF assembly” and “TFF system” are used interchangeably in this document and refer to a tangential flow filtration assembly in which fluid travels tangentially along the surface of the filter membrane within the filtration module of the system.

[0042] The terms “SPTFF component” and “SPTFF system” are used interchangeably in this document and refer to a single-pass tangential flow filtration system configured for single-pass mode operation, in which fluid passes through the system only once.

[0043] A “cassette” refers to a tube or plate filter element that contains stacked filtration (e.g., ultrafiltration or microfiltration) membrane sheets.

[0044] A "spiral-wound filter element" refers to a filter element that comprises a filter membrane and optionally spacer material wound around a central core. Spiral-wound filter elements are typically housed in a capsule.

[0045] "Capsule" refers to a filter element that contains a filter membrane or filter material matrix within a supportless tube.

[0046] "Hollow fiber filter element" refers to a filter element that contains a filter membrane bundle.

[0047] "Filtration membrane" refers to a selective permeation membrane used in filtration methods (e.g., TFF method, percolation method) to separate a feed into a permeate stream and a retained stream. Filtration membranes include, for example, but not limited to, ultrafiltration (UF) membranes, microfiltration (MF) membranes, reverse osmosis (RO) membranes, and nanofiltration (NF) membranes.

[0048] The terms "ultrafiltration membrane" and "UF membrane" are generally defined as membranes having a pore size ranging from about 1 nanometer to about 100 nanometers, or alternatively, by the membrane's "molecular weight cutoff," expressed in Daltons and abbreviated as MWCO. In various embodiments, the present invention utilizes ultrafiltration membranes having an MWCO rating ranging from about 1,000 Daltons to 1,000,000 Daltons.

[0049] As used in this article, the terms “microfiltration membrane” and “MF membrane” refer to membranes having pore sizes ranging from about 0.1 micrometers to about 10 micrometers.

[0050] The terms “liquid feed sample,” “feed,” “feed sample,” “liquid feed stream,” or “liquid stream” refer to the solution to be filtered that is delivered (e.g., continuously or in batches) to the filter module. The feed delivered to the filter module for filtration can be, for example, feed from a feed container (e.g., a vessel, tank) outside or inside the system, or retention material flowing from a pipe of an upstream filter module in the percolation system.

[0051] Additionally, the terms "liquid feed sample," "feed," or "feed sample," and "liquid feed flow" or "liquid flow" are used interchangeably to refer to a liquid feed sample or feed that flows from one container, device, etc., to another container, device, etc. Such movement is usually the result of pumping (e.g., by using a peristaltic pump or other types of pumps commonly known to those skilled in the art), but can also be achieved by other means, such as gravity.

[0052] As used herein, the terms “amount” and “amounts” refer to a non-limiting quantity of a sample (e.g., a liquid feed sample), measured, for example, by volume or weight. The terms are not limited to any particular volume or weight, etc. Nor are the terms limited to any particular unit of measurement. A term may refer to a portion of or the entirety of a specified quantity, i.e., a portion or the entirety of a liquid feed sample.

[0053] "Product" refers to the target compound in the feed sample. Typically, the product will be the biomolecule of interest (e.g., a protein), such as a monoclonal antibody (mAb). It may be present in the retention material or permeate, or both.

[0054] The term "total product volume" refers to an undefined volume equal to a production run, where "production run" is defined as the total amount or volume processed in succession or continuously. Total product volume is generally limited to the total volume available for a production run. It is undefined because different product runs may have different volumes depending on factors such as, for example, the product being produced (e.g., some products may have higher demand requiring larger production runs), the availability of raw materials (e.g., a limited supply of a particular medium may result in a smaller production run), or the capacity of the system used (e.g., a smaller processing system may be limited in the total volume that can be processed continuously or sequentially compared to a larger system).

[0055] The term "filtration" generally refers to the act of separating a feed sample into two streams, permeate and retention, using a membrane.

[0056] The terms "permeate" and "filtrate" refer to the portion of the feed that has permeated or filtered through the membrane. In other words, the portion of the feed flow that has passed through the membrane. These terms are used interchangeably.

[0057] The term "retention" refers to the portion of a solution that has been retained by a membrane, and retention can enrich the types of substances that have been retained.

[0058] A "feed line" or "feed channel" refers to a conduit used to deliver feed from a feed source (such as a feed container) or "sample source" to one or more processing units within a filtration assembly. The feed flow passes through the feed line or feed channel.

[0059] "Retainer line" or "retainer channel" refers to the piping in the filter assembly used to transport the retainer.

[0060] "Permeate line" or "permeate channel" refers to the piping in a filter assembly used to transport permeate.

[0061] When used in this document to describe processing units, the term "multiple" refers to two or more processing units (e.g., two or more filtering units).

[0062] "Flow-through connection" refers to the connection of two or more components of a filter assembly via one or more channels (e.g., feed channel, retention channel, permeate channel) that allow liquid to flow from one component to another.

[0063] "Processing" refers to the act of filtering (e.g., via TFF) a feed containing the product of interest, and then recovering the product in the form of concentration or purification.

[0064] "Purified" refers to a product prepared with a higher concentration of the desired component or product of interest and a lower concentration of contaminants compared to an unfiltered sample.

[0065] As used herein, "cycle tank" or "cycling tank" refers to a container constructed to store fluids and can be a single-use or reusable tank made of, for example, plastic, glass, or metal. It may also be capable of receiving or including an internal mixer to mix retained fluids, including, for example, conventional batch tanks, bags, and single-use bags. The cycle tank or recycling tank may also be integrally arranged in the feed line or feed channel, allowing it to capture a buffer volume of the feed, and need not be separate and distinct components. Preferably, the tank described herein exhibits good mixing for operational efficiency. This can be achieved, for example, by using an internal mixer and / or by designing the tank and retained fluid inlets to aid mixing. In percolation methods, the percolate can be added directly to the tank, but it is preferred to add it to the retained fluid line before introducing the combined fluids into the tank. This promotes mixing.

[0066] As used in this article, "pump" refers to a method or apparatus configured to induce flow (e.g., induce gravity flow) or apply a pressure differential to a fluid, including, for example, magnetic levitation pumps, peristaltic pumps, or diaphragm pumps.

[0067] "Transmembrane pressure drop" is the average pressure drop across the filter membrane module.

[0068] "Cross flow" is the velocity of the retained fluid between the inlet and outlet of the filter module. Unless otherwise specified, "cross flow" refers to the average cross flow velocity.

[0069] "Permeate flux" refers to the area-normalized flow rate of permeate in the permeate channels of the filtration module (e.g., L / m²). 2 / hr, LMH).

[0070] "Crossflow velocity" refers to the area-normalized average flow rate (e.g., L / m²) of the retained material in the feed channel of the filter module. 2 / min, LMM).

[0071] A "valve" is a method or component that stops or regulates fluid flow and / or redirects fluid flow through system piping. This can include physically moving pipes between tanks in an open system, "squeezing" pipes, on / off valves, control valves, and multiport valves.

[0072] The implementation plan for this example is described below.

[0073] Filtration systems and methods for continuous production In the biopharmaceutical industry, filtration has traditionally been performed as a batch process, where samples undergo several repeated filtrations, such as using TFF systems, or in a single pass, such as using SPTFF systems. Traditional batch filtration is an inherently discontinuous production method because the inflow and outflow of product from the filtration system ceases when the sample undergoes several repeated filtrations or when the process is stopped between batches for cleaning and / or sterilization. As the biopharmaceutical industry moves towards continuous processing of several unit operations (e.g., perfusion production and harvest, flow-through chromatography, single-pass concentration), there is a need for filtration methods and systems capable of providing continuous inflow and outflow of product. While in-line filtration can provide continuous inflow and outflow of product, such systems require undesirably large membrane areas, multiple tanks and buffer volumes, and become very difficult to operate in a cGMP environment to balance multiple flows. Therefore, there is a need for filtration systems and methods capable of performing filtration in continuous production processes that do not require undesirably large membrane areas, multiple tanks and buffer volumes, and are easy to operate.

[0074] Conventional batch filtration typically operates for only a few hours to minimize protein degradation from multiple pump passes, manage bioload, and time-balance the batch production pipeline to allow for pre- and post-treatment operational steps. The use of recirculation allows for continuous operation to balance the production pipeline, extends membrane utilization to reduce area requirements, and enables compact systems with sterile or disinfected components to manage bioload without adding significant complexity.

[0075] In the methods and systems of the present invention, filtration can be performed by circulating feed samples provided between two or more sample sources or feed sources. Thus, a total volume of multiple quantities is provided for filtration, allowing products to continuously flow into and out of the filtration system. Such a filtration system can be placed between any process or unit operation present upstream or downstream: for example, between SPTFF concentration, chromatography, clarification, and / or virus filtration unit operations. Such a multiplex filtration system can operate in series or parallel and can include combined products, such as chromatographic beads, cell substrates, or adsorbed polymers, wherein, if desired, the products are sequentially adsorbed, washed, eluted, and regenerated in a series of consecutive steps using membrane-assisted separation and retention.

[0076] An example of the filtration system 100 of the present invention is as follows: Figure 1The system may include a TFF system 150a, an SPFTT system 150b, or both in series. An exemplary circulation tank 110 is shown. One or more sample sources (not shown) are fed into the circulation tank. An optional storage tank 120 is shown. The feed is pumped from the circulation tank to the optional storage tank via pump 160a and / or from the circulation tank or storage tank to the TFF filtration system via pump 160b. A TFF filter 130 is also shown (RET = Retention; FEED = Feed from the storage tank or directly from the circulation tank; PER = Permeate). In some embodiments, optionally, a second circulation tank may be used, for example, in cases where a large volume of liquid feed samples is received or the first circulation tank requires cleaning or maintenance. The operator can then switch between circulation tanks as necessary or as required.

[0077] In one embodiment, permeate and / or retention are collected from the TFF filter until continuous filtration is complete. In the TFF system, at least a portion of the retention can be recycled to a storage tank or feed line if necessary to ensure the required filtration level. Continuous filtration is complete when the feed stream is exhausted (i.e., the total product volume has been processed). Optionally, buffer rinsing can be performed using a suitable buffer from buffer tank 180a. The choice of buffer depends on the product being processed and can be determined by those skilled in the art. Non-limiting examples include phosphate-buffered saline (PBS), Duchenne phosphate-buffered saline (DPBS), Hank's balanced salt solution (HBSS), HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid), and other suitable buffers known to those skilled in the art. In another embodiment, percolation can optionally be performed using a suitable buffer (DF) from percolation buffer tank 170. The choice of percolation buffer depends on the product being processed and can be determined by those skilled in the art.

[0078] In other embodiments, the filtration method can be an SPTFF filtration method. The feed can be pumped directly to the SPTFF system 150b from, for example, a circulation tank 110 (via pump 160c), or pumped to a storage tank 140 before the SPTFF system. In another embodiment, the feed can be a retention from the TFF system 150a. In one embodiment, the TFF system is used for percolation, while the SPTFF system is used for filtration. Again, the feed can be pumped directly to the SPTFF system from the circulation tank 110, thereby avoiding or completely eliminating the TFF system.

[0079] Similar to the TFF system, permeate and / or retention can be collected from the SPTFF system. Continuous filtration is complete when the feed stream is depleted (i.e., the total product volume has been processed). Optionally, buffer rinsing can be performed using a suitable buffer from buffer tank 180b. The choice of buffer depends on the product being processed and can be determined by those skilled in the art.

[0080] For clarity, the present invention can be operated as a TFF system, an SPTFF system, or a TFF system followed by an SPTFF system in series. For further clarity, the system can be operated as a filtration system or a percolation system. In another aspect, if the TFF and SPTFF systems are used in series, the TFF system can operate as a percolation system, and the SPTFF system as a filtration system. In yet another aspect, the TFF and SPTFF systems can operate individually or together first as a percolation system, followed by a filtration system, or as a filtration system followed by a percolation system.

[0081] Although not shown, the flow rate of the feed stream, retention material, permeate, and any pre- or post-operation fluids (e.g., flushing buffers, cleaning solutions, etc., see below) can be controlled and directed using appropriate valves. Flow rates can also be controlled by valve regulation (i.e., full or partial switching valves), either in conjunction with or separately from pump control. Flows from permeate tanks, buffer tanks, feed streams, circulation tanks, etc., can be controlled by valve regulation. Valve regulation (and pumps) can be controlled manually or via automated systems. Suitable automated systems are known to those skilled in the art.

[0082] Furthermore, one or more of the feed flow rate, retention flow rate, or permeate flow rate can be controlled. Control of various flow rates, either partially or entirely, throughout the system can be manual or automated. Automation can be achieved through computer-controlled systems or other devices such as mechanically controlled timers. Controlling one or more of the feed, retention, and / or flow rates can ensure that appropriate transmembrane pressure (TMP) is maintained for the filtration process being performed, depending on the filter type, feed material, and desired process outcome.

[0083] The system may optionally include pre-filtration and pre-percolation preparation components, as well as post-filtration and post-percolation cleaning components. For example, regarding pre-percolation preparation, containers for storing water (e.g., reverse osmosis deionized (RODI) water) and regeneration solutions (e.g., detergents, enzymes, acids, alkalis, etc.) are in fluid communication with a first buffer / regeneration valve that selectively directs the flow of buffer and regeneration solutions to the continuous filtration assembly. Second and third cleaning valves may also be included to direct and withdraw cleaning solution flows from the continuous filtration assembly if necessary.

[0084] In one embodiment, the system of the present invention includes a continuous filtration assembly, which is a tangential flow filtration (TFF) assembly. The TFF assembly may include a cartridge, a spirally wound filter element, a capsule, or a hollow fiber filter element. TFF provides an efficient method for filtration and, if desired, allows for sample concentration in the same unit operation. Therefore, TFF is advantageous for use in filtration methods.

[0085] In another embodiment, the system of the present invention includes additional TFF components located upstream and / or downstream of the filtration component to concentrate the sample or exchange buffer via percolation, for example, before / after filtration. If desired, such a TFF component can be a single-pass TFF component (SPTFF).

[0086] In embodiments, the filtration system of the present invention includes standard bioproduction equipment, such as conventional batch tanks, pumps, and control valves. The system also includes switching valves and / or perforated valves in fluid communication with the circulation tank and filtration assembly to allow the primary product stream to circulate between filling, filtration, and product recovery operations while continuously operating the filtration system (e.g., a TFF system).

[0087] The system and method of the present invention advantageously allow smaller amounts of product to undergo filtration while providing continuous production. By filtering smaller quantities, process time and therefore pump passes can be reduced compared to typical batch filtration processes. It is desirable to limit the number of sample pump passes, as biomolecules can be damaged during their journey through the pump and filtration equipment, leading to lower product yields and lower product quality. Furthermore, such a system and method can be used online in conjunction with other processing operations while also meeting the high buffer exchange requirements (if desired) for biological processing operations. By dividing the sample into multiple equal aliquots, smaller circulation tanks and filtration assemblies can be used, resulting in a smaller system footprint and shorter cycle times. Moreover, by sequentially running multiple product cycles by continuously operating filtration assemblies, the idle time between batches that occurs in conventional batch filtration is eliminated. With a continuously operating filtration system, the protein loading per membrane area can be increased, making it more economically feasible to discard the membrane at the end of the process, thereby eliminating washing time, washing solutions, and the need for reuse and revalidation, especially for large-scale operations.

[0088] In this implementation, sufficient time is allowed at the end of each filtration cycle to provide a buffer flush to recover larger volumes of product. For example, a buffer flush (also known as backwashing or rinsing) can be performed by introducing a buffer solution, air, or gravity discharge to push additional product located at or within the membrane of the filtration assembly or within fluid conduits / pipes back into the current circulation tank. The contents of the circulation tank are then provided as feed for subsequent unit operations. Alternatively, the sample can first be pumped out of the circulation tank and then progressively fed into a separate product collection tank with a buffer solution, subsequently providing its contents as feed for subsequent unit operations.

[0089] In a further implementation, sufficient time is allowed at the end of each filtration cycle to provide for a rapid regeneration cycle on the membrane, which can help maintain a constant filtration flux over extended cycle operations. The rapid regeneration cycle may not need to be performed after each filtration cycle, but can be invoked periodically or as needed.

[0090] In a further embodiment, after all filtration cycles, the system undergoes a complete or partial in-situ cleaning (CIP) procedure. Alternatively or additionally, all or some fluid piping and system components are replaced. For example, a TFF filter assembly including a disposable module can have such a module removed and discarded, or a circulation tank including a disposable bag can have such a bag discarded.

[0091] Control methods for filtration can be implemented through various control strategies. In one implementation, the continuous filtration process is controlled by monitoring and regulating TFF membrane crossflow and transmembrane pressure (TMP). TFF membrane crossflow can be controlled by adjusting the feed flow rate, retention flow rate, average crossflow rate, and / or the pressure drop setpoint of the feed pump. TMP can be controlled by a retention pressure control valve or by applying superimposed pressure to the circulation tank. During filtration, the recirculated product volume can be controlled to a constant setpoint (e.g., constant volume filtration) or a variable setpoint (e.g., optimal filtration) based on an algorithm that optimizes the concentration of product and / or buffer species in the circulation tank. Volume control can be monitored using a level probe or a pressure sensor associated with the circulation tank. Alternatively, the buffer addition flow rate and permeate removal flow rate can be measured using a flow meter, accumulator, and / or weighing scale. Such measurements can be provided to a controller that monitors the filtration process and regulates crossflow and TMP, for example, by switching valves or adjusting pump speed.

[0092] Endpoint control for filtration can also be achieved through various endpoint control strategies. In one embodiment, the filtration endpoint can be based on the cumulative volume of the added feed and / or the cumulative volume of the removed permeate, such as by measuring via a flow accumulator or weighing scale located at or near the fluid lines and tanks of the system, or by measuring via time-based measurements. In another embodiment, the filtration endpoint can be triggered based on a measurable property indicating that product treatment of the permeate or retained stream has occurred. For example, the filtration endpoint can be solution conditioning, such as a decrease in conductivity, where filtration is run prior to the next step, such as a TFF or ion exchange chromatography process, to reduce conductivity. This is especially true if the filtration method is a percolation method. Examples of measurable properties include pH, conductivity, refractive index, UV, turbidity, particle size measurements, or online or in-line near real-time direct measurements (e.g., determined by high-performance liquid chromatography (HPLC) or other analytical equipment) of the concentration of excipients, impurities, or target products.

[0093] Product recovery can also be achieved through various recovery strategies. In one embodiment, the flow rate and / or pump speed setpoint of the feed pump and / or filter pump are monitored. In a further embodiment, the tank recovery endpoint is monitored and controlled based on measurements from level, weight, and / or air sensors, measured volume accumulation, timed discharge duration, or other means of sensing that the tank is empty.

[0094] Finally, buffer rinse recovery can be controlled through various recovery strategies. In some implementations, buffer rinsing is monitored and controlled based on buffer pump flow rate, velocity, and / or discharge pressure. The endpoint of the buffer rinse can be triggered by rinse duration, accumulated volume, the weight of the buffer container in the recovery container, an air sensor, or other measurable properties of the protein / buffer solution, such as an ultraviolet (UV) signal.

[0095] In some embodiments, the continuous filtration system and method of the present invention may optionally also be provided for filtering relatively low molecular weight substances from other sample components. Examples of filtering permeable components relative to retained components include salts relative to proteins, proteins relative to nucleic acids, protein products relative to cells or flocs, viral and host cell proteins relative to cells, refolding reagents relative to proteins, alcohols relative to proteins, unreacted polyethylene glycol (PEG) relative to PEGylated proteins, unreacted toxins relative to antibody-drug conjugates (ADCs), and unreacted carbohydrates relative to conjugated vaccines.

[0096] The systems and methods of this invention may include filtration by microfiltration, ultrafiltration, reverse osmosis, or nanofiltration, depending on the molecules to be separated. Specific examples of suitable filtration membranes include Biomax®-30kD membranes and Ultracel®-30kD membranes (EMD Millipore, Bedford, MA) or other suitable membrane sizes for retaining the components of interest.

[0097] Examples of suitable buffer solutions for use in the systems and methods of the present invention, when required, include water, purified water, phosphate-buffered saline (PBS), acetate, and histidine. Example

[0098] Example 1: Reduction in membrane usage and system size The feasibility of continuous filtration was evaluated in this study, which tested two different methods and 12 different series (several feed and percolation concentrations). Method 1 utilized the cUFDF (continuous ultrafiltration / percolation) method of this invention, while the second method used a standard batch method (i.e., prior art methods) control scheme. The parameters tested were: 1) total membrane area (m²). 2 ); 2) Total membrane loading (kg / m³) 2 ); and 3) total tank volume (L). The series concentrations are given in Table 1.

[0099] Table 1-12 series have different inlet and percolation concentrations: (CbDF = concentration before percolation): Available Figure 2 , 3 As seen in section 4, compared to the standard batch process, the system using the method of the present invention requires a significantly lower total membrane volume, a significantly lower total tank volume, and a significantly increased total membrane load. Therefore, for the same production, the method of the present invention saves on membrane size and system size. Furthermore, it reduces the use of disposable equipment, as a single unit is typically used for longer process runs. Manual operation is also reduced, as frequent disconnections and reconnections are effectively eliminated.

[0100] Example 2. Conservation of production resources Another advantage of the method of the present invention is that it reduces the use of resources. Figure 5 The data shown presents typical process parameters for a day's production in a batch mode (open flow path), continuous UF / DF (open flow path), and continuous UF / DF (closed flow path).

[0101] Figure 6 The data shown illustrates the theoretical cost for the same three methodologies.

[0102] Example 3. Exemplary Continuous Filtering Method Procedure The crude product is collected in a recycling tank (e.g., Mobius Mix, EMD Millipore, Burlington, MA).

[0103] Once sufficient product has been collected, a continuous transfer to the TFF process system is initiated.

[0104] A TFF process system can be either an ultrafiltration or a diafiltration system.

[0105] The purified product is then returned to the SPTFF stage tank.

[0106] The product is concentrated by SPTFF, while the TFF system continues to process crude products from the production process by collecting crude products from multiple production sources in a circulation tank.

[0107] The rinsing and cleaning steps can be completed between filter cycles, for example, once a day or as needed.

[0108] All teachings from the patents, publications, and references cited in this article are incorporated herein in their entirety through citation.

[0109] Although exemplary embodiments have been specifically shown and described, those skilled in the art will understand that various changes in form and detail may be made without departing from the scope of the embodiments covered by the appended claims.

Claims

1. A method for filtering a liquid feed sample, the method comprising: (a) The first quantity of liquid feed sample is transferred from the first sample source to the circulation tank; (b) Simultaneously or in series with the first amount of liquid feed sample, the second amount of liquid feed sample is transported from the second sample source to the circulation tank; (c) When collecting a suitable amount of liquid feed sample in the circulation tank, the continuous feed stream of the liquid feed sample is transferred from the circulation tank to the tangential flow filtration (TFF) system to filter the continuous feed stream of the liquid feed sample. (d) During the filtration of liquid feed samples, one or more additional amounts of liquid feed samples are delivered to a circulation tank to maintain a continuous feed flow of liquid feed samples to the TFF system; (e) Continue to supply liquid feed samples to the circulation tank until the total product volume is processed by the TFF system.

2. A method for filtering a liquid feed sample, the method comprising: (a) The first quantity of liquid feed sample is transferred from the first sample source to the circulation tank; (b) When collecting a suitable amount of liquid feed sample in the circulation tank, the continuous feed stream of the liquid feed sample is transferred from the circulation tank to the tangential flow filtration (TFF) system to filter the continuous feed stream of the liquid feed sample. (c) During the filtration of liquid feed samples, one or more additional amounts of liquid feed samples are delivered to a circulation tank to maintain a continuous feed flow of liquid feed samples to the TFF system; (d) Continue to supply liquid feed samples to the circulation tank until the total product volume is processed by the TFF system.

3. The method of claims 1 and 2, wherein the additional amount of liquid feed sample is derived from one or more different sample sources.

4. The method according to any one of the preceding claims, wherein the TFF system is a single-pass tangential flow filtration (SPTFF) system.

5. The method according to any one of the preceding claims, wherein the liquid feed sample is concentrated by filtration.

6. The method according to any one of the preceding claims, wherein the liquid feed sample is a cell culture medium.

7. The method according to any one of the preceding claims, wherein the liquid feed sample comprises a monoclonal antibody.

8. The method according to any one of the preceding claims, wherein the liquid feed sample is concentrated before being conveyed to the circulation tank.

9. The method according to any one of the preceding claims, wherein the liquid feed sample is diluted before being delivered to the circulation tank.

10. The method according to any one of the preceding claims, wherein the filtration is percolation, and wherein the method further comprises a buffer tank in fluid communication with the feed stream.

11. The method according to any one of the preceding claims, the method further comprising a storage tank between the circulation tank and the TFF system.

12. The method according to any one of the preceding claims, the method further comprising rinsing the TFF system after the total volume of processed product has been reached.

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